A low-power, wide-dynamic-range integrated humidity sensing chip is implemented using a printable polymer sensing material with an on-chip pulse-width-modulation interface circuit. By using the inkjet printing technique, poly/polystyrene sulfonate that has humidity sensing features can be printed onto the top metal layer of a 0.35 μm CMOS IC. The developed printing-on-chip humidity sensor achieves a heterogeneous three dimensional sensor system-on-chip architecture. The humidity sensing of the implemented printing-on-chip sensor system is experimentally tested. The sensor shows a sensitivity of 0.98% to humidity in the atmosphere. The maximum dynamic range of the readout circuit is 9.8 MΩ, which can be further tuned by the frequency of input signal to fit the requirement of the resistance of printed sensor. The power consumption keeps only 154 μW. This printing-on-chip sensor provides a practical solution to fulfill an ultra-small integrated sensor for the applications in miniaturized sensing systems. AmongM networkTo achieve above features, gas-sensitive polymer materials are a good choice for the low-power requirements because polymer sensors can be operated without heaters to reduce the power consumption ,12. In aIn this paper, we propose a heterogeneous integration platform for gas-sensing, which could implement a small-form-factor and a low-power system with ease. Using the additive characteristics of the inkjet printing process, a pseudo-3D integrated circuit was prepared. Using this method, the circuit area could be vertically combined with the sensor device. A readout CMOS chip was specifically designed for inkjet printing integration. This simple pulse-width-modulation (PWM) readout architecture also has the potential for extending it to multi-sensor array topologies.2.2.1.4 to 106 Ω). As a consequence, the interface circuit needs to adapt to the variation of the printing process. To overcome this issue, we choose a wide-dynamic-range and low-power readout circuit to implement the integrated readout circuits [R), the frequency, and the duty cycle of input signals. In this design, we set the input frequency as 32.768 kHz, which can be simply implemented by a crystal oscillator. The CR is designed as 2 pF. With these conditions, the designed dynamic range is Rmax = 9.8 MΩ; Rmin ∼ 500 kΩ; the resolution is 1.5 ns/kΩ.The resistance of the printed sensor could respond to the gas concentration over a huge range  × 10 μm (L) gap. This circuit also fulfilled low-power requirement, the simulated average power consumption is 135 μW, without full-digitized conversion block. The simulated full function power consumption is 400 μW.The sensor interface chip was designed and fabricated on a 0.35 μm CMOS Bio-MEMS platform, which includes a gold layer post-process step. The gold layer is vital for connecting the polymer sensor device because gold has good compatibility with conductive polymers . After t2.3.x [A gas sensing material, we chose poly/polystyrene sulfonate (PEDOT:PSS), a widely used conductive polymer, as the backbone ingredient. PEDOT:PSS composite materials could sense many kinds of gases, such as water , NOx [22x . The aqux ,25. Prisx . The sma2.4.2, NO and CO tests, these testing gases were pre-mixed with N2 in different concentrations. The mixed gases were flushed into the chamber for 10 min. After 10 min, the chamber was evacuated by vacuum pump and flushed with N2 for 10 min. This sequence was repeated several times to complete the sensitivity test. For the humidity sensing tests, in addition, the test gas was composed of N2 pumped through a buffer tank with heated water. Tuning the ratio of water vapor and dry N2 can control the humidity of the chamber. For the ethanol (EtOH) sensing test, a specific volume of ethanol was injected into the inlet by a microsyringe pump and flushed into the chamber with dry N2. All the sensor testing conditions were at a pressure of 1 atm under room temperature. The input signal and the supply voltage (3 V) were applied by a National Instrument data acquisition card (USB-6363). Moreover, the output signals were also captured by the same equipment. To monitor the humidity profile in real-time, a precise hygrometer, which has a built-in SHT11 (Sensirion) sensor chip, was installed in the chamber as a reference.Each sensor was tested in a customized sealed chamber under different gas conditions. For CO3.versus resistance of the sensor. The discrepancy in the pre-simulation and post-simulation results is caused by the parasitic capacitances and resistances. This offset can be linearly calibrated after CMOS fabrication. For a 32 kHz, 50% duty cycle input signal, the maximum readout range is limited in 9.8 MΩ. The dynamic range can be adjusted to a higher value by simply altering the input duty cycle, or by decreasing the input frequency.After the inkjet printing post-process, the sensor-integrated chip was tested under different kinds of gas conditions. The experimental data of the humidity-sensing test is shown in 2, NO, CO, and EtOH were tested. The experimental results are shown in 2, CO, and NO, the sensors showed relatively weak responses at low concentrations. For the stability test, the two kinds of sensors were stored in an electronic dry box under 30% RH. The measured results are shown in To test the sensitivity and selectivity of PEDOT:PSS and PEDOT:PSS/AZO sensors against different gases, four kinds of gases including COThe specifications of the implemented chip and a comparison with the state-of-the art designs for similar purposes are summarized in 4.2. The small-form-factor and low-power integrated sensor chip shows the potential to achieve multi-sensors on a single chip for M2M networks applications in IAQ monitoring or healthcare.A prototype of printed humidity sensor-on-chip is presented. Implemented by an inkjet printing technique, polymer-based gas sensing materials were 3D stacked on the top of CMOS circuits. The CMOS chip is fabricated by the TSMC 0.35 μm BioMEMS 2P4M CMOS process, and the inkjet printing is accomplished using a self-made inkjet printer. With a low power consumption of 154 μW from a 3 V supply, the experimental results demonstrate that the sensors have good sensitivity for humidity and alcohol. The core chip area is only 0.5 × 0.57 mm


The SGNP with greatest sensitivity was tested using clinical samples in Indonesia in 2013–2014. As a result, the novel method was able to detect low concentrations of DENVs using only 6 μL of serum, with similar sensitivity to that of a Qiagen RNA extraction kit using 140 μL of serum. In addition, this method allows for multiplex-like identification of serotypes of DENVs. This feature is important for good healthcare management of DENV infection in order to safely diagnose the dangerous, highly contagious disease quickly, with high sensitivity.We propose a novel method of detecting trace amounts of dengue virus (DENVs) from serum. Our method is based on the interaction between a sulfated sugar chain and a DENV surface glycoprotein. After capturing DENV with the sulfated sugar chain-immobilized gold nanoparticles (SGNPs), the resulting complex is precipitated and viral RNA content is measured using the reverse-transcription quantitative polymerase chain reaction SYBR Green I (RT-qPCR-Syb) method. Sugar chains that bind to DENVs were identified using the array-type sugar chain immobilized chip (Sugar Chip) and surface plasmon resonance (SPR) imaging. Heparin and low-molecular-weight dextran sulfate were identified as binding partners, and immobilized on gold nanoparticles to prepare 3 types of SGNPs. The capacity of these SGNPs to capture and concentrate trace amounts of DENVs was evaluated  Dengue viruses (DENVs), which have 4 phylogenetically and antigenically distinct types (DENV-1 to DENV-4), are emerging arthropod-borne flaviviruses that cause dengue fever (DF), dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS), mostly in tropical and subtropical countries . A largeCurrent DENV diagnostic methods are based on serological detection of circulating antibodies. Specificity of these tests suffer due to cross-reactivity from antibodies against related flaviviruses, thus they are insufficient. Alternative methods for the detection of DENV rely on the measurement of viral RNA using reverse transcription polymerase chain reaction (RT-PCR). RT-PCR –12 has sd-glucan [Our innovative approach is based on exploiting the interaction of a DENV envelope protein (glp E) with sulfated sugar chains. It was reported that dengue virions comprise 180 identical copies of envelope proteins, which cover the viral surface , 17. Infd-glucan .in vitro. Finally, we compared our novel SGNP method with a commercially available RNA extraction kit to detect DENV in patient serum and plasma samples in a clinical setting using reverse-transcription quantitative PCR (RT-qPCR).Here, we describe a novel method to capture and concentrate DENVs by sugar chain-immobilized gold nanoparticles (SGNP), followed by PCR-based detection. First, we identified DENV-binding sugar chains to be used for capture and concentration of DENV. Second, the selected sugar chains were immobilized on gold nanoparticles to prepare SGNPs , 25. ThiEthical approval (63/PT02.FK/ETIK/2010) was provided by the Ministry of Health of the Indonesian Government, and written informed consent was obtained from all human subjects. All samples were a part of the Community Base Dengue Study (CBDS) conducted by the Indonesia University Cipto Mangun Kusumo Hospital. The samples were stored at −70°C until analysis.DENVs  were kindly provided by Drs. Kase and Hiroi at Osaka Prefectural Institute for Public Health. DENVs were cultivated in freshly cultured Vero cells.A freshly prepared and purified DENV solution from the culture supernatant of DENV-infected Vero cells was diluted (1:20) with PBS-T. These diluted DENV samples were run through the SPR device at a flow rate of 150 μL/min. After 60 s, the buffer was changed to PBS-T with the same flow rate for 3 min to analyze dissociation. Binding was monitored using the CCD camera in the SPR imaging system . After each run of 1 sample, the chip was washed with 10 mM NaOH for 2 min and re-equilibrated with PBS-T for 5 min.4, with stirring, at 100°C. The heparin-linker conjugate or the dextran sulfate-linker conjugate was then added to the solution with gentle stirring, and the solution was stirred for 1 h at 25°C in the dark. The solution was centrifuged at 12,000 × g for 40 min to obtain a precipitate. After several washes with double-distilled water (ddH2O), the precipitate was re-suspended in ddH2O and adjusted to an optical density (A530) of 0.5 using a cuvette with a 1-mm long light path, to obtain Hep-cGNP or DS25-cGNP.Based on the results from SPR imaging, and due to their common availability, heparin and low-molecular-weight dextran sulfate were selected and immobilized on gold nanoparticles to prepare Hep-cGNP and DS25-cGNP, respectively. Two conjugation methods were used, as previously reported , 20, 21,4 with stirring at 4°C. The linker-dextran sulfate conjugate was then added to the solution, and incubated with gentle stirring for 1 h at 4°C in the dark. The solution was dialyzed against ddH2O using a dialysis membrane with MW 3500 cutoff and then lyophilized. The lyophilized powder was re-suspended in ddH2O and adjusted to the concentration of 10 mg/mL. Sequential centrifugation was performed at 12,000 × g for 15 min to obtain the precipitate. The precipitate was re-suspended in ddH2O and adjusted to 1 mg/mL with ddH2O to obtain the DS25-GNP solution.As an alternative to produce low-molecular-weight dextran sulfate-immobilized gold nanoparticles, 50 mM of sodium tetrahydroborate was added to 1.3 mM NaAuCl2O was measured by dynamic light scattering . On average, Hep-cGNP particles had a diameter of 60 nm, DS25-cGNP a diameter of 48 nm, and DS25-GNP a diameter of 27 nm.The concentrations of heparin and low-molecular-weight dextran sulfate were measured using the p-aminobenzoic acid ethyl ester method . Heparin4 or 1:106 with PBS and mixed with 10 μL of SGNP. This mixture was incubated at room temperature with shaking for 30 min. The mixture was then centrifuged  to separate the precipitate. The precipitate was washed with PBS and treated with 20 μL of RNase-free water containing 0.1% SDS to disrupt virus particles, and then centrifuged again at 10,000 ×g for 10 min at 4°C to remove SGNPs.Five hundred micro-liters of the DENV-1 culture supernatant was diluted 1:10g for 10 min at 4°C) to separate the precipitate from the supernatant. The precipitate was washed with PBS and treated with 20 μL of RNase-free water containing 0.1% SDS to disrupt viral particles. The mixture was centrifuged again at 10,000 × g for 10 min at 4°C to remove SGNPs.Six micro-liters of a patient’s serum or plasma was mixed with 600 μL of PBS at room temperature. Five hundred micro-liters of the above solution was then mixed with 10 μL of DS25-GNP and incubated at room temperature with shaking for 30 min. The mixture was then centrifuged  according to the manufacturer’s instructions. RNA was eluted in 40 μL of the supplied elution buffer.et al [Two micro-liters of eluate from the Qiagen kit elution, as well as the precipitate-derived RNA solution, the separated supernatant, and the original solution without SGNP were used as templates for real-time PCR amplification. The primers were prepared according to Poovoraman et al  , 100 μL of serum was added into the sample well of the device. The test results were recorded after 15–20 min. Similarly, in the SD Bioline IgG/IgM test, 10 μL of serum was added into the sample well of the device. We then added 4 drops (~120 μL) of the assay diluent to the round-shaped assay diluent well. The results were recorded after 15–20 min. The test results were analyzed and interpreted according to the manufacturer’s instructions.The interaction between various sugar chains and DENVs was evaluated using a surface plasmon resonance (SPR) imaging system . To select a high-affinity sugar chain for effective capture of DENVs, we used a gold-coated chip containing 48 types of sugar chains immobilized on the surface see . A cultu4 and 1:106 in PBS) were used as virus input, and the results of the capture are shown in m value of DENV-1 to be 84.7–85.1°C. All SGNPs tested captured and concentrated DENV-1 from the sample that had been diluted 1:104. However, at the lower concentration (1:106), we only detected efficient capture of DENV-1 virions with DS25-GNP. Due to the greater sensitivity and efficacy, we used DS25-GNP for further testing.Three types of SGNPs  were synthesized as previously reported , 30; we 4 to 1:106 1% human serum in PBS. The diluted DENVs were captured and concentrated on DS25-GNP and then quantified using RT-qPCR-Syb. DENVs were detected in the precipitate from all DS25-GNP samples, even at the 1:106 dilution (4). These results clearly indicate that DS25-GNP captured and concentrated DENV-2 and -4 in the presence of 1% serum, even at low DENV concentrations.To evaluate the ability of DS25-GNP to perform under more physiologically relevant conditions, we next tested the ability of DS25-GNP to capture and concentrate DENV virions in the presence of serum. We prepared DENV-2 and -4 suspensions from cell culture supernatants, and diluted the samples 1:10et al. reported that RT-PCR could be used to discriminate between serotypes of DENVs [m values for all DENVs tested were too similar to one another; therefore, serotyping based on the Tm value alone was problematic. On the other hand, our results from experiments performed in Indonesia using a Bio-Rad PCR apparatus, but otherwise under the same conditions, indicate that we were able to determine the DENV serotypes based on their specific Tm values. To compare our SGNP method with the gold standard method [Before we evaluated our detection technique using serum samples from dengue patients, we tested whether our RT-qPCR assay could be performed using protocols previously validated . Purifieof DENVs . Howeverd method , we testThe protocol used for clinical research was approved by the Ethics Committee of the Indonesia University, Faculty of Medicine, Department of Microbiology, and the protocol was registered under the number 63/PT02.FK/ETIK/2010. This research was performed according to the Declaration of Helsinki on Human Rights. To determine the efficacy of our new protocols in a clinical setting, we tested serum samples from 87 patients, which were collected from 4 geographic areas in Indonesia: Pangkal Pinang, Lampung, Jakarta, and Solo. Except for the samples from Pangkal Pinang, 65 samples were additionally assayed using IgG-, IgM-, NS1-, and PCR-based methods. All samples were collected in the acute phase of disease (days 1 to 5 after presentation of the first fever).We compared the efficacy of isolating DENV RNA by SGNP capture with a commercially available kit: the QIAamp Viral RNA Mini Kit from Qiagen . Six microliters of patient serum or plasma was diluted 100 times with PBS, and 500 μL of the diluted solution was mixed with 10 μL of SGNP. The mixture was incubated, and SGNP collected by centrifugation. The precipitate was washed with 500 μL of PBS and resuspended in 20 μL of a 0.1% SDS solution to release RNA from DENV. In parallel, RNA was isolated from 140 μL of the same patient’s serum or plasma using the Qiagen QIAamp kit. RNA from patient samples was extracted and bound to the filter according to the manufacturer’s instructions. Finally, the bound RNA was eluted with 40 μL of the supplied elution buffer. All RNA-containing solutions, which included re-suspended samples of the precipitate, supernatant, diluted serum/plasma without SGNP treatment, and the Qiagen kit-eluted solution, were analyzed by RT-qPCR-Syb.RNA isolated from the serum and plasma samples of 87 patients using both our SGNP concentration protocol and the Qiagen QIAamp kit were used as templates for our RT-qPCR-Syb protocol. We detected signal for DENV from 25 SGNP samples, and 23 Qiagen kit-eluted samples . Among tOverall, our SGNP method was effective in detecting DENV RNA from only 6 μL of serum or plasma. Using our protocols, we were able to determine that the serotypes of the SGNP-positive samples collected in Pangkal Pinang were DENV-1, -2, and -3. Those from Solo were all DENV-4. It is noteworthy that SGNP-isolated RNA and Qiagen kit-isolated RNA resulted in the detection of different DENV serotypes.Rapid, early, and highly sensitive methods for DENV detection are being actively developed and improved due to the worldwide prevalence of DENVs, the high risk of DENV infection (such as severe dengue (DHF/DSS)) in some geographic areas, and because of the absence of both a vaccine and an effective drug against DENV –15. We am curves. Our results from clinical testing demonstrated that all subtypes of DENV were detected in Solo, Lampung, and Pangkal Pinang. In a few cases, the subtype of DENV detected in the re-suspended SGNP precipitate was different from that detected in the Qiagen kit-eluted RNA from the same serum sample. The reason for this discrepancy is unclear, but one possible explanation is the difference in the methods used to isolate viral RNA. The re-suspended SGNP precipitate is derived from intact viral particles, whereas the Qiagen kit-eluted RNA represents chemically extracted total RNA/DNA from the serum sample. If this total RNA/DNA contains RNA from destroyed DENV particles previously involved in infection, and which released RNA into plasma/serum, then the above discrepancy may occur. By contrast, our SGNP method provides information related to intact DENV virions, which are the infectious particles. Therefore, the results obtained using the re-suspended SGNP precipitate likely provide information that is more relevant to medical treatment than the Qiagen RNA-extraction method.After concentration of DENV, we selected the RT-qPCR-Syb method for detection of viral RNA. We chose this method because PCR-based methods have higher sensitivity and accuracy compared with antibody-based methods, such as the assays for NS1, IgG, or IgM. In addition, the use of the intercalator SYBR Green I was expected to work well in a multiplex-like assay for identification of the subtypes of DENV in a single reaction by carefully comparing the TOur method is based on the interaction between specific proteins on the surface of the viral particle with low-molecular-weight dextran sulfate on the surface of SGNPs. In the blood of patients with a history of DENV infection, viral particles may interact with DENV-specific antibodies. It was reported that IgM and IgG react with epitopes present on the envelope protein , 34, 35.et al. [in vivo studies using mice deficient in type I and II IFN receptors.Most severe cases of dengue occur via primary infection in infants whose mothers have some DENV immunity, and secondary heterotypic infection in children and adults through ADE. According to the ADE model, the viral burden may increase due to the presence of non-neutralizing antibodies. Infection via ADE alters the innate and adaptive intracellular antiviral mechanisms, resulting in unrestricted viral replication and an increase in the serum viral load . This moet al.  reportedet al. . Thus, det al. . Howeveret al. . TherefoS1 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableYellow cells indicate the corresponding Tm values for each of serotype. Red cells indicate the severe status of patients. Red ovals indicate positive results.(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


S-selective hydroxynitrile lyase (S-HNL) activity, which we call designed S-HNLs; these proteins folded as efficiently as the native S-HNL. Sequence and biochemical analysis of the designed S-HNLs suggested that accumulation of neutral mutations occurs during the process of S-HNLs evolution from a low-activity form to a high-activity (native) form. Taken together, our results demonstrate that our software and the associated methods could be applied not only to design of complete sequences, but also to predictions of protein evolution, especially within families such as esterases and S-HNLs.Development of software and methods for design of complete sequences of functional proteins could contribute to studies of protein engineering and protein evolution. To this end, we developed the INTMSAlign software, and used it to design functional proteins and evaluate their usefulness. The software could assign both consensus and correlation residues of target proteins. We generated three protein sequences with  To date, several methods for enzyme design have been reported, including ancestral sequence designTo perform consensus design, one must first assign the consensus residues, i.e., the residues that appear most commonly at each position in the aligned sequences of proteins in the family of interest89Although designing full-consensus proteins has certain advantages, creation of these proteins requires overcoming several challenges. First, the library should be prepared by selecting large numbers of sequences that are not phylogenetically biased, because biased libraries often generate proteins with low activities relative to the native proteins91414S-selective hydroxynitrile lyases (S-HNLs). S-HNLs catalyze the degradation/synthesis of cyanohydrins, including (S)-mandelonitrile, to/from aldehyde and cyanide ionS-HNL is suitable as a target protein for validation of INTMSAlign for several reasons: i) S-HNL sequences could not be designed without curation, because the library contained more sequences of esterases than S-HNLs. ii) Many structural and biochemical analyses of S-HNLs have been performed previously18192021S-HNL is also an industrially useful protein23S-HNL activity; therefore, we refer to them as designed S-HNLs. We analyzed the biochemical functions of the designed S-HNLs by measurement of CD spectra and enzyme kinetics. Our results show that INTMSAlign could assign both consensus and correlation residues, and that this software could be applied to the design of functional proteins. Finally, we will consider the protein evolution of the S-HNLs based on experimental data obtained from our designed S-HNLs and previous researches performed by other groups25As mentioned above, assignment of consensus and correlation residues is necessary for design of functional proteins. To date, however, few algorithms capable of finding these residues have been reported. To address this need, we developed the MSA software INTMSAlign. We then investigated whether the program could assign these residues in sequences of the α/β hydrolase fold superfamily, which includes esterases and Ntrial ofiles are created. The ofile is the input file for INTMSAlign; each file contains one STP and Npick sequences selected randomly from the library  was used as the STP, and the consensus residues were extracted from the library. MeHNL is a native S-HNL, and many functional analyses of this protein have been performed previously. S-HNL, which belongs to the α/β hydrolase fold superfamily, catalyzes addition and elimination of a nitrile group from its substrates28MeHNL and the esterases is relatively high: for example, the identity between MeHNL and salicylic acid binding protein 2 (SABP2) esterase from tobaccoIn this study, the sequence of MeHNL to the Blastp web server  was 1000, and the number of sequences extracted from the library (pickN) was 8 (MeHNL (Ser80 and His236) as consensus residues (The runtime parameters of INTMSAlign are shown in b server . Becausek) was 8 . From thresidues .MeHNL. In a previous study, Asano et al. showed that mutation of these residues could improve the solubility of MeHNL in an E. coli expression systemMeHNLE. coli expression system can be solubilized by mutating certain residues to the consensus residues.At this point, an important question arises: what is the advantage of assigning consensus residues when we perform protein engineering? To illustrate the advantage of such assignments, we investigated the consensus residues at positions 103, 176, 199, and 224 of Collectively, these results confirm that INTMSAlign can assign consensus residues.Sequences belonging to families other than that of the STP were frequently included in INTMSAlign libraries, and the existence of such sequences made it difficult to assign consensus residues from a single family. Curation of the library is necessary in order to resolve this difficulty; therefore, a function called “residue fixation” was implemented to INTMSAlign . An overMeHNL, enzyme sequences from at least two families were present in the library: esterases and S-HNLs. In this case, the library contains more esterase sequences than S-HNL sequences, i.e., the library is phylogenically biased toward esterases. Thus, curation of the library is required to assign consensus residues specific to the S-HNL family. A previous study chose position 237 as the marker residue; this amino acid is Met in esterases and Lys in S-HNLsS-HNLs . From comparison of both files, we detected five residues that are individually conserved in esterases and S-HNLs  could be expressed in E. coli. HNL85 and HNL54 were expressed in the soluble fraction, and more than 5 mg of proteins were obtained from 1 L of culture. On the other hand, the solubility of HNL30 was lower than that of the other two designed S-HNLs: less than 0.5 mg of the protein could be obtained from 1 L of culture.Three of designed S-HNLs were measured to confirm whether the S-HNLs could fold correctly  of HNL85, HNL54, and HNL30 were 60, 67.5, and 50.5°C, respectively. Thermal renaturation also occurred in the designed S-HNLs; about 60% (HNL85 and HNL54) and 40% (HNL30) of the protein were refolded by lowering temperature  of HNL54 and HNL30 were, respectively, about 0.01- and 0.5-fold of that of MeHNL - and (S)-Man as substrates -Man grew as a function of the amount of enzyme - and (S)-Man -Man (S)-Man was more than 10-fold higher than that of HNL54 (R)-Man was about 0.5-fold of that of HNL54 -Man -Man . Among t (R)-Man . The catof HNL54 , whereasof HNL54 . Ultimatn > 100, . The higreaction .S-HNL activity could be ranked in the following order: HNL85 < HNL54 < HNL30. Despite the presence of mutations in active-site residues specific to S-HNL, the S-HNL activity of HNL85 was too weak to determine the kinetic parameters Taken together, the level of rameters  and 3. Irameters  could swS-HNLs revealed that their catalytic efficiencies (kcat/Km) toward rac- and (S)-Man increased in the following order: HNL85, HNL54, and HNL30 .Enzyme kinetics of the designed nd HNL30  and 3. IS-HNLS-HNLs (residues 115–147 and 178–186). Because flexibility and conformational change in this region are important for the substrate specificity of S-HNLsS-HNLs may affect their enzyme kinetics  was located within 10 Å of the Oγ atom of Ser80, which is a catalytic residue of kinetics  and 3. TS-HNLs, the remote locations of the mutation sites were confirmed. Therefore, localization and dynamic behavior during the reaction are the main factors that affect the enzyme kinetics of the designed S-HNLs.In other enzymes, such as simvastatin synthaseS-HNLs from sequence and performed biochemical analysis of designed S-HNLs. These analyses demonstrate that the software we developed, INTMSAlign, can be utilized to predict evolution. The results of these predictions led us to postulate that the evolution of esterases ultimately resulted in native-like S-HNL activity. This hypothesis was based in part on the observation that both S-HNLs and esterases share high sequence and structural similarity with each other. Furthermore, in contrast to the S-HNLs, whose existence is only confirmed in certain cyanogenic plants, esterases with α/β hydrolase folds are present in a wide range of species30S-HNLs would evolve in the following order: HNL85, HNL54, HNL30. A schematic model of the predicted protein evolution of the S-HNLs is shown in In this study, we predicted protein evolution of S-HNL via the following steps: A) Trade-off between esterase activity and S-HNL activity -Man would increase, as would the activity toward (S)-Man (S)-Man is elevated. The second process corresponds to the evolution from HNL54 to HNL30. Here, a functional trade-off occurred during conversion from HNL54 to HNL30; S-HNL activity was improved by sacrificing enzymatic efficiency toward (R)-Man -Man . This pr (R)-Man . In addiof HNL30 .S-HNLs to achieve high activity and selectivity. In general, enzymes attain new functions by mutating residues in the core regions, such as in step A, but the function is often quietly lower than that of native enzymes. In addition, the mutation often destabilizes the enzyme37S-HNLs, HNL54 is the most stable, and is therefore most able to accommodate an unfavorable mutation in the core region.During protein evolution from HNL85 to HNL30, mutations mainly occurred at positions remote from the active site, such as on the protein surface . This suS-HNL, as determined by the analysis of the designed S-HNLs. Alternatively, an ancestral esterase or S-HNL might exist, and it might attain esterase or S-HNL reactivity via various genetic processes, such as gene duplication. In addition, the predicted model  software was developed to assign both consensus and correlation residues. INTMSAlign is derived from the well-known MSA program CLUSTALWhnl85 gene encoding designed S-HNL (HNL85) was synthesized and ligated to plasmid pIDSMART by Integrated DNA Technologies . The resultant plasmid, pIDSMART-hnl85, was digested with NdeI and XhoI and electrophoresed; the digested hnl85 gene was extracted from the agarose gel and ligated into the expression vector pET28b . This plasmid was used to transform E. coli strain BL21(DE3). Transformants were cultivated for 4–5 hrs at 37°C in 1 L of LB medium supplemented with 30 μg/mL of kanamycin. To induce protein expression, 1.0 mM isopropyl-β-D-thio-galactopyranoside (IPTG) was added to the broth, and the culture was grown for an additional 24 hrs at 20°C.The S-HNLs (HNL54 and HNL30).After cultivation, cells were collected and suspended in buffer A (20 mM potassium phosphate [pH 7.0] and 100 mM NaCl) containing 10 mM imidazole, disrupted by sonication, and centrifuged. The supernatant was loaded onto a Ni-Sepharose column, and the column was washed with 50 ml of buffer A containing 70 mM imidazole. The sample was then eluted with buffer A containing 300 mM imidazole. The eluate was purified on a gel-filtration column  using buffer B (10 mM potassium phosphate [pH 7.0] containing 50 mM NaCl) as the loading buffer. All purification procedures were performed at 4°C. The purity of the sample was checked by SDS-PAGE. Protein concentration was determined by monitoring absorbance at 280 nm. An identical procedure was applied to the other designed R)-, or (S)-mandelonitrile . Reaction mixture - or (S)-Man) was incubated at 30°C for 30 min, and then transferred to a cuvette. After the enzyme solution was added to the cuvette, measurement of time-dependent absorbance change at 280 nm using a UV-2600 UV-Vis spectrometer  was initiated immediately and proceeded for 1.5 min. The production rate of benzaldehyde (ε280 = 13800 M−1 cm−1) was calculated using the ORIGIN software . All experiments were conducted in triplicate.Initially, HNL activity was measured by monitoring benzaldehyde formed by degradation of mandelonitrile, e.g., racemic, (R)- and (S)-Man. One hundred microliters of purified S-HNL was added to 900 μl of reaction mixture consisting of 300 mM citrate (pH 4.2), 100 mM KCN, and 50 mM benzaldehyde, and the mandelonitrile synthesis reaction was allowed to proceed at 30°C for 5 min. After the reaction was complete, 100 μl of the reaction mixture was removed and added to 900 μl of organic solvent (n-Hexane:2-Propanol = 85:15). Ten microliters of the solvent was then applied to a CHIRALCEL OJ-H column equipped on a the UFLC Prominence Liquid Chromatograph LC-20AD HPLC system , and the UV-absorbance at 254 nm was monitored. The retention times for benzaldehyde and (R)- and (S)-Man were about 5.4, 11.5, and 14.4 min, respectively. The details of the experimental method were described previously20Second, we analyzed the cyanohydrins synthesis reaction. Activity was measured by monitoring synthesis of (S-HNLs were measured utilizing a Jasco J-715 CD spectrometer. The buffer contained 10 mM potassium phosphate (pH 7.0) and 50 mM NaCl, and 0.06 mg/ml of purified designed S-HNLs was utilized in the measurement. Far-UV spectra were recorded from 195 to 280 nm. Wavelength scans recorded ellipticity every 0.5 nm.Circular dichroism (CD) spectra of the designed For thermal denaturation and renaturation, the ellipticity change at 222 nm was recorded. Temperature was increased from 5°C to 95°C, and then continuously decreased from 95°C to 5°C. Data were collected in 0.5°C steps. The temperature increased at 60°C/hr. The fraction of folded protein was calculated using the following equationtθ is the observed ellipticity at any temperature, Fθ is the ellipticity of the folded form, and Uθ is the ellipticity of the unfolded formHere, α is the fraction folded, Supplementary information


The suboccipital muscles are connected to the upper cervical spinal dura mater via the myodural bridges (MDBs). Recently, it was suggested that they might work as a pump to provide power for cerebrospinal fluid (CSF) circulation. The purpose of this study was to investigate effects of the suboccipital muscles contractions on the CSF flow. Forty healthy adult volunteers were subjected to cine phase-contrast MR imaging. Each volunteer was scanned twice, once before and once after one-minute-head-rotation period. CSF flow waveform parameters at craniocervical junction were analyzed. The results showed that, after the head rotations, the maximum and average CSF flow rates during ventricular diastole were significantly increased, and the CSF stroke volumes during diastole and during entire cardiac cycle were significantly increased. This suggested that the CSF flow was significantly promoted by head movements. Among the muscles related with head movements, only three suboccipital muscles are connected to the upper cervical spinal dura mater via MDBs. It was believed that MDBs might transform powers of the muscles to CSF. The present results suggested that the head movements served as an important contributor to CSF dynamics and the MDBs might be involved in this mechanism. The suboccipital area consists of complex anatomical structures. The myodural bridge (MDB) as dense soft tissue communication extends from the suboccipital musculatures to the cervical spinal dura mater123456789101112131412345789101345787810714The cardiac cycle-related CSF flow pulsations were depicted as CSF flow waveforms. Based on the waveforms, the CSF flow quantitative parameters of the craniocervical junction were calculated. And the results of both scans and the comparison between them are presented in The temporal CSF dynamics parameters were presented in the The CSF dynamics amplitude parameters were presented in the The CSF dynamics volumetric parameters included the mean values of the systolic CSF flow volume (VS), the diastolic CSF flow volume (VD) and the net-flow volume   and werep value, 0.002).Furthermore, the detailed CSF stroke volume variations after the one-minute-head-rotation period were analyzed and results were presented in p value 0.320).In the above results, among forty cases, a small number of measurements were found as outliers based on Box and Whisker Plots analysis. It was impossible to re-measure the subjects with outliers, so these outliers were not included in T-test. Additionally, the intervals between the one-minute-head-rotation and the second cine-PC scan ranged from 1.0–3.6 minutes, with a mean value of 1.9 ± 0.57 minutes, because of the process of re-positioning MRI scan ahead of the second cine-PC scan. During the scanning procedure, the heart beat of each subject was monitored. The mean heart rate values before and after the head rotation period were of 70.7 ± 8.8 and 69.9 ± 7.3 beats per minute, respectively, showing no significant change in heart rate after the head rotation period , including most researchers involved in this study, was subjected to MR imaging . Informed consent was obtained from all of them and none of them had any history of cardiovascular, neurological, endocrine and cervical disorders.2; section thickness, 5 mm; and 2 signal intensity average. The encoding direction was in the head-to-foot orientation for all volunteers. PC- images were obtained at each time points, for a total of 25 measurements equally distributed over the cardiac cycle. The imaging time duration varied between 2 to 4 minutes, depending on the volunteer’s heart rate.A cine-PC MR imaging methodBefore the cine-PC scan, sagittal T2 weighted images of head and neck were obtained by a quick MR scan to provide anatomic details. Based on the median sagittal images, a transverse plane for the cine-PC scan was designated at the level of the atlas upper border a.Each volunteer was scanned twice. The first scanning was performed after a resting period. After that, each volunteer was required to perform continuous head rotations for a period of 60 seconds, while lying on their back on the MRI bed and their heads against the bed. The head rotation speed was set at around 0.5 cycles per second. One head rotation cycle was counted when the head was rotated from median position to one side and next by another side and then return. Volunteers turned their heads according to the researcher’s instructions for normalization of the one-minute-head-rotation. A scan for re-positioning underwent immediately after the one-minute-head-rotation period and then the second cine-PC scan followed.Cine-PC images were transferred to an independent workstation (aw46mr) for CSF flow analysis using the Analyze (Report Card 4.0) software. Using the region-of-interest function of the Analyze software, an irregular contour was drawn manually to encompass the entire cross-sectional area of the subarachnoid space at the level of the atlas upper border . The regp value of less than 0.05 indicated a statistically significant difference.The range, mean, and standard deviation were calculated for each parameter. All parameters were compared between both scans acquired before and after the one-minute head rotation period. Statistical significance was calculated using a paired sample t-test or Chi-Square test. A How to cite this article: Xu, Q. et al. Head movement, an important contributor to human cerebrospinal fluid circulation. Sci. Rep.6, 31787; doi: 10.1038/srep31787 (2016).


NAc precursor to yield UDP-MurNAc-pentapeptide. MurD catalyzes the addition of D-glutamic acid to UDP-MurNAc-L-Ala in the presence of ATP; structural and biochemical studies have suggested the binding of the substrates with an ordered kinetic mechanism in which ligand binding inevitably closes the active site. In this work, we challenge this assumption by reporting the crystal structures of intermediate forms of MurD either in the absence of ligands or in the presence of small molecules. A detailed analysis provides insight into the events that lead to the closure of MurD and reveals that minor structural modifications contribute to major overall conformation alterations. These novel insights will be instrumental in the development of new potential antibiotics designed to target the peptidoglycan biosynthetic pathway.The biosynthetic pathway of peptidoglycan, an essential component of bacterial cell wall, is a well-recognized target for antibiotic development. Peptidoglycan precursors are synthesized in the bacterial cytosol by various enzymes including the ATP-hydrolyzing Mur ligases, which catalyze the stepwise addition of amino acids to a UDP-Mur N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues linked by β1→4 bonds. The lactoyl group of the MurNAc residues is substituted by a peptide stem most commonly composed of L-Ala-γ-D-Glu-meso-A2pm (or L-Lys)-D-Ala-D-Ala . Cross-linking of the stem peptides generally occurs between the carboxyl group of D-Ala at position 4 and the amino group of the diaminoacid at position 3, either directly or through a short peptide bridge. [The development of bacterial resistance to antibiotics worldwide is a phenomenon that urges the need to discover new antibacterial drugs. The biosynthetic pathway of peptidoglycan, a key component of the bacterial cell wall, is a tractable target since proteins that catalyze key reactions in the pathway can be targeted both by natural and synthetic antibiotics. –4 Peptid bridge. N-acetylmuramic acid (UDP-MurNAc), thereby building a stem peptide. Mur ligases require ATP for activity, and are absolutely essential for bacterial survival. [The biosynthesis of peptidoglycan can be roughly divided into three stages: cytoplasmic, membrane-associated, and periplasmic. Within the bacterial cytoplasm, Mur ligases perform the stepwise addition of amino acids to UDP-urvival.  The mechurvival. –21 Theseurvival. –13From a structural point of view, Mur ligases are three-domain enzymes that share a very well conserved ATP-binding site that harbors the Gly-X-X-Gly-Lys-Thr/Ser , 14 motiNAc-L-Ala, generating the dipeptide moiety L-Ala-D-Glu in almost all bacterial species. [E. coli MurD reveals two open conformations: an unbound one and an UDP-N-acetylmuramoyl-L-alanine (UMA)-complexed one. [E. coli MurD has been the object of targeted molecular dynamics simulation studies that have shed light on the conformational changes that occur upon ligand binding as presented in E. coli MurD has been the object of extensive studies aiming at the development of new inhibitors, [E. coli MurD either in the presence or in the absence of ligands. This work led us to identify novel conformational properties of MurD involving an intermediate conformation in the presence of ADP and UMA, as well as an intermediate conformation in the absence of ligands. This work thus reveals that substrate binding is not the strict causative agent of domain closure, and that Mur enzymes display a variable amount of flexibility, both in the presence and absence of substrates, which could be essential for their activity in the cell. In addition, our structural analyses indicate that the kinetic mechanism of MurD, which had previously been suggested as being ordered by similarity with MurC and MurF [MurD catalyzes the addition of D-glutamic acid to UDP-Murspecies.  As mentixed one.  In additxed one. –9, 16 E.ibitors, –43 but oibitors, , 42–43 oibitors, , 44–45 Iand MurF , 13 may 2+-nitrilotriacetate-agarose column and elution with a discontinuous gradient of imidazole. The enzyme was recovered in the 100 mM fraction. It was dialysed against 20 mM Hepes (pH 7.4), 200 mM NaCl, 5 mM DTT, 0.05% (w/v) NaN3. The amount of protein obtained was determined by the Bradford method, quantitative amino acid analysis, and by measuring the absorbance at 280 nm. The purity of the protein was checked by SDS-PAGE and MALDI-TOF mass spectrometry. [MurD was expressed and purified using the 6×His-tag system described. DH5α cells harbouring the pABD16/MurD vector were grown in 2-YT medium containing ampicillin (100 μg/mL) at 37°C in a rotary shaker to reach A600nm of 3.5. Expression was induced by adding isopropyl β-D-thiogalactopyranoside at a final concentration of 1 mM and growth was continued overnight at 20°C. The cells were lysed by sonication and the 6×His-tagged protein was purified by affinity binding to a NiIntermediate Free MurD):Crystallization of the intermediate conformation of ligand free MurD  and 2 μL of reservoir solution (1.8 M (NH4)2SO4, 7% (v/v) (±)-2-methyl-2,4-pentanediol and 0.1 M MES, pH 5.6) at 15°C by vapor diffusion using the hanging-drop method. Crystals grew in 48 hours. X-ray diffraction data were collected at the European Synchrotron Radiation Facility  ( France) .Intermediate Bound MurD):Crystallization of intermediate conformations of MurD with ligands  with 2 μL of reservoir solution . Crystals appeared in 6 months and data was collected at the ESRF, as above  split iry: 3uag  The soluRP/wARP.  COOT [49RP/wARP.  was usedRP/wARP.   and Intermediate Bound MurD .Structures of  enzyme. –10 Sincei) Intermediate Free MurD and ii) Intermediate Bound MurD are both compared to the previously solved structures of MurD, and iii) similarities and differences between the two new conformations of MurD are highlighted.Results regarding these structures are presented in three separate sections in which Intermediate Free MurD, 421 residues were refined. There is one minor region missing between residues Thr180─Tyr187 since it could not be traced in the electron density map. Notably, Lys198 is not carbamoylated as it is the case in open MurD structures.7In the solving of Intermediate Free MurD structure, we superposed it onto that of other previously solved E. coli MurD structures IntermediIntermediate Free MurD is different from all of the orientations found in the aforementioned structures of MurD , [2+ [2+ [Intermediate Bound MurD structure by superimposing it to the previously solved structures (Intermediate Bound MurD with open UMA-bound MurD [d to UMA  Previous (UMAG),  UMA-ADP-ructures . Superpoructures  revealedructures . Furtherructures . It appeund MurD  Fig 4C)IntermediIntermediate Bound MurD structure, the H-bond pattern of UMA binding is almost the same as in the closed structures of MurD [Intermediate Bound MurD . [Intermediate Bound MurD represents the conformation which is adopted after substrates are bound, but it is not yet in the closed “active” conformation, since Lys198 is not carbamoylated. So far, there was no evidence that carbamoylation of Lys198 is a transient event. Nevertheless, the crystal structures of open MurD , and closed MurD , suggest that carbamoylation of Lys198 occurs during catalysis. Of course, the possibility of chemical degradation of KCX198 due to the long period in which the crystals grew cannot be totally ruled out.However, the importance of carbamoylated Lys198 (KCX198) must not be neglected. KCX198 was only found in closed “active” conformations of MurD.  Its impoet al. , who estrescue).  ThereforIntermediate Bound MurD displays some features of the closed conformation of MurD. The hinge loop is similar to the one in open conformation of MurD, but with an additional H-bond to Pro300 and ADP, thus dictating the C-terminal domain to take a position closer to central domain than open conformation of MurD. [in vacuum were on average between 10 and 15 kcal/mol. [5–6 kcal/mol for the bond in vacuum and 0.5–1.5 kcal/mol for the bond in aqueous solution, [of MurD.  However,cal/mol.  The estiolution,  which isolution,  which giolution,  With totin vivo in the form of a multi-enzyme complex; [Caulobacter crescentus [Mycobacterium tuberculosis [Thermotoga maritima [Another aspect of the biology of Mur ligases is their assembly inside the bacterial cytoplasm. It has been speculated that Mur ligases are assembled complex;  however,escentus , Mycobacrculosis  and Thermaritima . In the maritima  Whether The kinetic mechanism of the Mur ligases was firmly established in the cases of MurC  and MurFThe crystal structures presented in this paper provide us with new tools in the urgent mission to discover novel antibacterial agents. So far, inhibitors of MurD that were designed to bind in the substrate-binding sites have not expressed significant antibacterial activity. , 22 TakiIntermediate Free MurD): 5a5e (Intermediate conformation of ligand free MurD (D): 5a5e .Intermediate Bound MurD): 5a5f (Intermediate conformation of MurD with ligands (D): 5a5f .S1 Checklist(PDF)Click here for additional data file.S2 Checklist(PDF)Click here for additional data file.


The precursor to copper transformation was performed and annealed at 600°C with the shape preserved. The copper micro-rods are assembled from unique thin lamellar layers, each with the thickness of approximately 200 nm and nano-pores of approximately 20 to 100 nm. This morphology is highly related to the crystal structure of the precursor. The mechanism of the morphology formation is proposed, which would be able to offer a guideline toward porous metals with controllable macro/micro/nano-structures by the precursor crystal growth and design.Porous metallic copper was successfully prepared by a simple thermal decomposition strategy. A coordination compound of Cu(BTA)The online version of this article (doi:10.1186/s11671-015-0769-7) contains supplementary material, which is available to authorized users. Porous metallic materials have become a burgeoning field in both applied technology and basic scientific research, especially for their significant thermal or electron conductivity, catalysis properties, importance in interface engineering, energy industry, and biomedical applications -4. Durin2 (BTA: bis[1(2)H-tetrazol-5-yl]amine) as the precursor was synthesized, based on the following merits. First, among various coordination compounds that contain high-nitrogen ligands, Cu(BTA)2 is mildly energetic during combustion process, which is favored for both the reaction and the porous structure formation  direction, the Cu-Cu distance is as large as 10.2 Å. Therefore, the Cu(BTA)2 presents a layered crystal structure. This layered structure matches well with the layered structure of the corresponding copper. From this clue, the mechanism of the copper micro/nano-structure formation is suggested. The decomposition of each Cu(BTA)2 molecule generates a single Cu0 atom at the position in situ. These isolated Cu0 atoms are highly active to attract each other forming a Cu-Cu metallic bond. The Cu-Cu metallic bond has the length of 3.6 Å, smaller than the Cu-Cu distance in the precursor. So the Cu0 atoms would preferably approach from the short distance in a statistical thermodynamic way, that is, in the (100) crystal plane of the precursor. Thus, it intends to form a Cu layer structure with the thickness in one-atom scale. However, as the Cu-Cu bond formed, it obeys the lowest energy rule to transform into the fcc phase instantly. These initially formed Cu nano-clusters undergo alloying to form bigger crystals. The degassing process during the decomposition may contribute to the layered nano-structure: the decomposed N2- and carbon-containing gases would escape preferably through the gaps between the layers, which may generate oriented pressure to shape the metallic copper.To understand how this orientation is formed, we take a closer look at the Cu(BTA)e Figure . The Cu and lower than pure copper (5.96 × 107 S∙m−1) [6 S·m−1, which is one order of magnitude higher than that of the Cu micro-rod. It indicates that the electrical property depends not only on the component fractions, size, and shape but also on the porous structure: when intensively pressed, the pores are eliminated, leading to the enhancement of the electrical conductivity [The electrical conductivity of one Cu micro-rod was measured at room temperature Figure . The ele7 S∙m−1) , which i7 S∙m−1) . In compuctivity . The micuctivity -33, sensuctivity , and catuctivity .Figure 52 crystal as the precursor. The shape of the crystals was preserved for the copper product. This allows us to obtain copper with various morphologies by the growth of the precursor crystals of different sizes and shapes, without changing the molecule itself. Moreover, the layered nano-structure is highly related to the crystal parameters of the precursor. It could be expected that the same precursor crystallizes in different crystalline spaces; accordingly, the micro/nano-structure would be tuned.In summary, we report a thermal decomposition method to prepare copper micro-rods with layered porous structure for the first time, by using a well-designed coordination compound of Cu(BTA)


Background: Some drugs of abuse down regulate the expression of cystine/glutamate (xCT) antiporter in the nucleus accumbens (Acb) after extinction or withdrawal. The altered level of xCT exchanger in Acb, a structure involved in ethanol reinforcement, may contribute to the pathological glutamatergic signaling, linked to addiction. We hypothesized that the expression of xCT may be changed in Acb and whole brain also in non-dependent , ethanol-dependent rats, as well as, during ethanol withdrawal.Methods: Wistar rats were made ethanol-dependent by chronic exposure to an alcoholic milk beverage (from 2.4 to 7.2% v/v ethanol). Ethanol non-dependent rats were exposed to a similar, but non-alcoholic liquid diet and self-administered ethanol (10%) twice a week. Withdrawal in ethanol-dependent rats was studied at 12 h after the last ethanol-enriched diet exposure. Immediately after the measurement of somatic signs of withdrawal, Western blot analysis with a polyclonal antibody against xCT was carried out in a naïve control group, non-dependent and ethanol-dependent rats as well as withdrawal rats, in order to study the level of xCT expression in Acb and whole brain.Results: Non-dependent rats self-administered an average dose of 1.21 ± 0.02 g/kg per session (30 min). Daily ethanol consumption during chronic exposure to the alcoholic beverage ranged from 6.30 ± 0.16 to 13.99 ± 0.66 g/kg. Ethanol dependent rats after suspension of the ethanol-enriched diet have shown significant somatic signs of withdrawal. Western blotting analysis of Acb lysates revealed that xCT was over expressed in ethanol-dependent rats whereas in whole brain preparations xCT was over expressed in both non-dependent and ethanol-dependent rats compared to control group. On the contrary, xCT expression during withdrawal was down regulated in Acb and restored to control level in whole brain preparations.Conclusions: The changes of xCT expression in both Acb and whole brain following ethanol dependence and withdrawal indicate that xCT might represent a novel therapeutic target for the treatment of ethanol addiction. The development of ethanol dependence is posited to involve numerous changes in brain neurotransmission that lead to characteristic physiological signs upon abstinence from ethanol.Increased glutamatergic neurotransmission appears to mediate the reinforcing properties of ethanol and changes are considered responsible of affecting many aspects of neuroplasticity associated with ethanol dependence  among which the Slc7a11 gene, has been shown to be expressed at higher levels (1.54-fold), whereas other authors have shown that ethanol, dose-dependently, increases the xCT exchanger expression in mouse hepatic stellate cells  and to the Ministry of Health , and in strict accordance with the European Council directives on the matter [n. 2007/526/CE]. All possible efforts were made to minimize animal pain and discomfort and to reduce the number of experimental subjects.Ethanol solutions (v/v) obtained by dilution of ethanol  , weighing 175–225 g at the beginning of the experiment, were housed in pairs in Plexiglas cages. The colony room was maintained under controlled environmental conditions  under a 12-h light/dark cycle .Male Wistar rats (Four groups of rats were utilized for this study:n = 8). These rats were fed the non-alcoholic liquid diet.Ethanol non-dependent rats allowed to self-administer ethanol twice a week . These rats were fed the alcoholic liquid diet.Ethanol dependent rats (n = 17) after suspension of the ethanol-enriched diet.Ethanol dependent withdrawn: rats (n = 6).Control rats that was used only for xCT measurements, fed the non-alcoholic liquid diet  with (dependent) or without ethanol (non-dependent). Non-dependent rats  drank for 30 min (twice a week) the “desired” amount of ethanol. Instead, the dependence was induced by “forced” ethanol liquid diet  or without (control and ethanol non-dependent) ethanol fresh whole cow milk, 910–970 ml /l;ethanol 25–75 ml/l;vitamin A 5000 IU/lsucrose 17 g/l.This mixture (with or without ethanol), freshly prepared daily, according to the method of Uzbay and Kayaalp , supplieTraining was conducted in modular operant chambers, located in ventilated soundproof environmental cubicles (Med Associates Inc. USA). Each chamber was equipped with a non-retractable drinking cup (capacity 0.50 ml) and two nose-poke holes located 3 cm to the left and right of the cup. A white light placed above the active hole and an orange light placed above the inactive hole were used as environmental stimuli. Only the active nose-poke hole set off the dipper-delivering solution (0.1 ml) into the drinking cup in 3.05-second period. Explorations at both the active and inactive nose-poke holes were recorded. In particular, recording at the inactive hole served to control for specificity of the response in the operant chamber. The availability of liquid was signaled by a house light placed on the wall in front of the drinking cup that would light up for the duration of liquid delivery. Following each delivery, there was a 2-second time-out period during which responses had no consequences and the white light placed above the active hole went off. An infrared head detector was located in the reservoir and recorded all signals during the entire session. The chambers were interfaced to a computer equipped with software that ran the programmed sessions and recorded the data. For operant ethanol (5–10% v/v) self-administration, rats were trained to nose-poke under a fixed-ratio 1 (FR1) schedule of reinforcement, in which each response resulted in 0.1 ml of solution delivery. From day 1 to day 6, rats were permitted to nose-poke explore for 5% ethanol solution. Starting on day 7, the ethanol percentage was gradually increased, with daily increases of 1% up to the final concentration of 10%. Following the acquisition, after a stable baseline of responding was reached, operant self-administration of ethanol was then increased to a FR2 schedule until the self-administration behavior was stable and subsequently, the schedule requirement was increased to a FR3 , 4.8% (4 days) and 7.2% (14 days) ethanol. This diet composition and regimen has been reported to result in a significant correlation between ethanol-containing liquid diet consumption and blood ethanol level after 21 days of treatment, as reported by Kayir and Uzbay . Rat's bWithdrawal in ethanol-dependent rats was studied at 12 h after the last ethanol-enriched diet exposure. Withdrawal behavioral signs were determined exactly at 12 h after ethanol suspension. Each subject was placed under white light conditions in Plexiglas observation chambers (25 × 20 × 25 cm) and observed for 5 min by an observer blind to the subject's treatments. The following somatic signs of withdrawal were recorded: body tremors (BT), tail rigidity (TR), vocalization (VOC) and ventro-medial limb retraction (VmLR). We used a rating scale adapted from Macey et al.  as folloTo measure anxiety-like responses upon ethanol withdrawal, the elevated plus maze (EPM) test was used. The test was performed immediately, after 12 h after ethanol suspension. The apparatus consisted of 2 gray Plexiglas open arms and 2 black enclosed arms , with similarly shaped arms opposite to each other. The 5-min test procedure began when the animal was placed in the center of the maze, facing an open arm. The time (min) spent in open arms and the number of open arm entries were scored and used as measure of anxiety-like behavior  and whole brain sections  were suspended in saline and subsequently homogenized as described below.Immediately after the behavioral observations, rats were intra-peritoneally injected with 1.3 g/kg of ethylic urethane . Under deep anesthesia, rats were sacrificed and brains removed and immediately frozen at −80°C (4 h) before being sectioned. Brains were rapidly dissected into coronal sections on an ice-cooled metal plate using a scalpel. The brain regions were identified according to the rat brain atlas . Homogenization was carried out with a Dounce homogenizer using 20 strokes of the loosely fitting pestle. The lysates were sonicated with an ultrasonic homogenizer  for 20 s on ice at 20% amplitude, and centrifuged at 12,000 × g for 15 min at 4°C. The protein content of supernatants was measured according to Bradford , 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM NaBradford , using bhttp://imagej.nih.gov/ij/).Aliquots of protein lysates (80 μg) were separated on 12% SDS gel Laemmli,  and tranU-test. Numbers of open arm entries were analyzed by two-way ANOVA. Time spent in open arms were analyzed by one-way ANOVA. In the presence of overall significant main effects and interactions , the Least Significant Differences (LSD) post-hoc test was performed.All values are expressed as mean (± s.e.m.). Ethanol intake values (in dependent rats) were analyzed by one-way ANOVA. Rat body weights were analyzed by two-way ANOVA (repeated measure). The 4 different withdrawal signs  were assessed by individual comparison among individual means using the non-parametric Mann-Whitney t test, assuming a p < 0.05 as statistically significant. A power analysis for t-test with the free software G*Power 3.1 (http://www.gpower.hhu.de/) was also carried out. The results of this analysis gave a power (1-β err prob = 0.95) with a sample size of 3 and an α level of 0.05. Being the power higher than 0.80  of 3 different experiments ± s.e.m. and analyzed by unpaired Student's Non-dependent rats, allowed to self-administer ethanol (10%) twice a week, self-administered an average dose of 1.21 ± 0.02 g/kg per session (30 min).F = 127.18, p < 0.0001].Daily ethanol consumption during chronic exposure to the alcoholic beverage (from 2.4 to 7.2% v/v ethanol) ranged from 6.30 ± 0.16 to 13.99 ± 0.66 g/kg. One-way ANOVA revealed a significant increase in ethanol consumption along the exposure time , time  and a significant group x time interaction  revealed that the alcoholic beverage was responsible of a significant loss of body weight in ethanol-dependent rats.Average body weights in ethanol-dependent rats showed a progressive decrease from the beginning to the end of the study compared with ethanol non-dependent groups. However, significant main effects of group , of open-closed/session entries  but not a significant treatment x open-closed/session entries interaction  indicating a significant difference for entries into open arms between withdrawal vs. ethanol non-dependent group (p < 0.0001) and ethanol dependent group and a significant difference for entries into total arms between withdrawal vs. ethanol non-dependent group (p < 0.0001). In addition, there was a non-significant difference between withdrawal and ethanol-dependent group both into open arms and into total arms entries  = 11.86, p = 0.00014] indicated a significant difference for time spent into open arms between ethanol non-dependent vs. ethanol-dependent (p = 0.0015) and between ethanol non-dependent vs. withdrawal groups (p < 0.0001)  Figure .p < 0.05) and non-dependent rats (p < 0.05). Following 12 h withdrawal in dependent rats, the level of xCT expression with respect to control (p < 0.05), non-dependent (p < 0.05) and ethanol-dependent (p < 0.05) samples, was significantly down regulated. The results of 3 experiments, expressed as A.U., are shown in Figure The expression of xCT in Acb and whole brain homogenates was investigated by Western blot analysis. The antibody recognizes a band of about 40 kDa, which corresponds to the more active xCT isoform . The level of protein was even more increased in brain of rats dependent from ethanol, and this increase was significantly different from control group (p < 0.05) and ethanol non-dependent (p < 0.05). Following withdrawal, the level of xCT expression was restored to the level of control group . On the other hand, the xCT expression was strongly decreased in Acb homogenates of ethanol-dependent animals after 12 h withdrawal. These changes in xCT expression could be responsible for alterations in glutamate levels in the Acb that may occur in response to chronic ethanol exposure and that may contribute to the pathological glutamate signaling linked to withdrawal-induced behavior. The profound down-regulation in the expression of xCT in the Acb, following 12 h of withdrawal in dependents rats could be also linked to the recidivism for ethanol abuse after a period of abstinence  have higher levels of xCT expression than control group, and a further significant increase of xCT in ethanol-dependent rats, in which xCT expression was reduced to control values after 12 h withdrawal. These data suggest that an intermittent , or a long-term ethanol exposure (in the liquid diet), cause an increase of the xCT in the whole brain. Interestingly, these results are in agreement with data obtained from Lin et al. , who repThe activity of xCT contributes to the maintenance of a cellular redox balance, sufficient to protect cells from oxidative damage  could be useful for the management of ethanol withdrawal symptoms. xCT is also over-expressed in the whole brain homogenates from both non-dependent and ethanol dependent rats and restored to control level following ethanol withdrawal, probably as a consequence of the brain oxidative stress induced by ethanol exposure and by ethanol deprivation in withdrawal, respectively. Overall, these findings indicate that xCT expression is altered in both the Acb and the whole brain. xCT might therefore represent a novel therapeutic target for the treatment of ethanol abuse.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


V3.1, CaV3.2 and CaV3.3 mRNA expression levels were assessed in the hippocampus and amygdala using qPCR. Z944 was not effective at suppressing seizures in fully kindled rats compared to vehicle. Animals receiving Z944 required significantly more stimulations to evoke a class III (p<0.05), IV (p<0.01) or V (p<0.0001) seizure, and to reach a fully kindled state (p<0.01), than animals receiving vehicle. There was no significant difference in the mRNA expression of the T-type Ca2+ channels in the hippocampus or amygdala. Our results show that selectively targeting T-type Ca2+ channels with Z944 inhibits the progression of amygdala kindling. This could be a potential for a new therapeutic intervention to mitigate the development and progression of epilepsy.Temporal lobe epilepsy (TLE) is the most common form of drug resistant epilepsy. Current treatment is symptomatic, suppressing seizures, but has no disease modifying effect on epileptogenesis. We examined the effects of Z944, a potent T-type calcium channel antagonist, as an anti-seizure agent and against the progression of kindling in the amygdala kindling model of TLE. The anti-seizure efficacy of Z944  was assessed in fully kindled rats (5 class V seizures) as compared to vehicle, ethosuximide  and carbamazepine (30mg/kg). Each animal received the seven treatments in a randomised manner. Seizure class and duration elicited by six post-drug stimulations was determined. To investigate for effects in delaying the progression of kindling, naive animals received Z944 (30mg/kg), ETX (100mg/kg) or vehicle 30-minutes prior to each kindling stimulation up to a maximum of 30 stimulations, with seizure class and duration recorded after each stimulation. At the completion of drug treatment, Ca Temporal lobe epilepsy (TLE) is the most common epilepsy syndrome that is resistant to drug treatment . Most caThe kindling model has been used extensively as a functional model of TLE. In kindling, repetitive stimulation results in a progressive increase in the severity and duration of the seizures , 5. Once2+) levels with the development of seizures  and thus no longer observed at the longer time point assessed here. We did note a non-significant trend for increased mRNA expression of CaV3.1 and CaV3.3 in the amygdala and in all three T-type Ca2+ channels in the hippocampus in the kindled + vehicle group.An increase in T-type Cal of TLE , however2+ channel blocker, Z944, is effective in delaying seizure progression in the amygdala kindling model. This may suggests that pharmacologically targeting T-type Ca2+ channels may be an effective disease modifying strategy to inhibit the onset or progression of TLE. However, further testing in other TLE models should be performed before the anti-epileptogenic role of Z944 is established.The results of this study provide evidence that the selective, high potency, T-type Ca


Using validated measures of individuals’ perceptions of their neighborhood built environment is important for accurately estimating effects on physical activity. However, no studies to date have examined the factorial validity of a measure of perceived neighborhood environment among older adults in the United States. The purpose of this measurement study was to test the factorial validity of a version of the Abbreviated Neighborhood Environment Walkability Scale (NEWS-A) modified for seniors in the Nurses’ Health Study (NHS).A random sample of 2,920 female nurses (mean age = 73 ± 7 years) in the NHS cohort from California, Massachusetts, and Pennsylvania completed a 36-item modified NEWS-A for seniors. Confirmatory factor analyses were conducted to test measurement models for both the modified NEWS-A for seniors and the original NEWS-A. Internal consistency within factors was examined using Cronbach’s alpha. The hypothesized 7-factor measurement model was a poor fit for the modified NEWS-A for seniors. Overall, the best-fitting measurement model was the original 6-factor solution to the NEWS-A. Factors were correlated and internally consistent.This study provided support for the construct validity of the original NEWS-A for assessing perceptions of neighborhood environments in older women in the United States. A growing body of built environment research has focused on associations between older adults’ perceptions of their neighborhood environment and physical activity -4. HowevVarious measures of the perceived neighborhood environment have been used across different studies. One commonly used measure of individuals’ perceptions of their neighborhood environment is the Neighborhood Environment Walkability Scale (NEWS) and its abbreviated form (NEWS-A) -4,8-10. There may be important aspects of the neighborhood environment with respect to walking for older adults, which the commonly used NEWS and NEWS-A and less commonly used single items may not measure. For example, pedestrian and personal safety features of the neighborhood environment  may be particularly salient to older adults with conditions that have impacted their balance, visual acuity, and physical reaction time. Therefore, the purpose of this study was to develop and test the factorial validity of a modified NEWS-A for seniors in a sample of older women in the United States.Participants were selected from the Nurses’ Health Study (NHS) cohort, which started in 1976 with 121,701 married nurses aged 30 to 55 years living in 11 of the most populous U.S. states [In the biennial NHS questionnaire, participants reported their birth date , race , ethnicity , highest level of education , current living situation , and walking limitations . In the 2008 supplemental questionnaire, participants reported length of residence at current address .The modified NEWS-A for seniors that was tested in this study consisted of seven subscales with 36 items. The original six subscales (24 items) from the original NEWS-A were included  . Nine ada priori 7-factor solution to the modified NEWS-A for seniors was tested using confirmatory factor analysis (CFA) based on a variance-covariance matrix. CFA was also conducted for the six original subscales in the original NEWS-A instrument [Frequency tables and logistic regression were used to examine predictors of missing data for all items in the modified NEWS-A. An strument . We inita priori model fit was unacceptable, models were re-specified based on a review of factor loadings, modification indices, and conceptual meaningfulness. All analyses were conducted in 2012 using SAS 9.3 for UNIX.Model fits were assessed by root mean square error of approximation (RMSEA <0.06) , root meParticipants’ ages ranged from 61.5 to 88.4 years with a mean of 73.0 ± 6.9 years. The participants were mostly White (97.3%) and non-Hispanic (99.2%). The majority (66.9%) had a registered nurse (RN) degree only, 27.6% had a bachelor’s, master’s or doctoral degree, and 5.5% were missing information on education. Participants in California were slightly older than participants in Massachusetts and Pennsylvania (75.6 years vs. 71.8 and 71.6 years). A slightly lower percentage of participants in California were Caucasian compared to that in Massachusetts and Pennsylvania (93.1% vs. 99.8% and 99.2%). Education levels were lowest in Pennsylvania (76.3% had RN degree only), followed by Massachusetts (68.3%), and California (56.5%). Participants were similar in demographics  to the full cohort (data not shown).Overall, less than 3% of the data at the case level in the modified NEWS-A for seniors were missing. Results of logistic regression analyses examining missing data points in relation to age, race, ethnicity, and education suggested there were no significant associations between demographic characteristics and missing data (corrected α .01 for multiple tests). Therefore, confirmatory factor analyses were conducted using full information maximum likelihood estimation with data from the final analytic sample of 2,920 participants. Furthermore, confirmatory factor analyses were tested using both full information maximum likelihood and list-wise deletion for missing data. Results were substantively similar; therefore, only models using full information maximum likelihood are reported.a priori 7-factor solution to the modified NEWS-A for seniors did not adequately fit the observed data  and the finding that a parsimonious walkability scale, the original NEWS-A, has factorial validity for use among older women in three U.S. states. In conclusion, the six subscales of the original NEWS-A appear valid for measurement of how older women perceive their neighborhood walking environments.


In the forced swimming (FST), the immobility times in both OVX and Sham-LPS groups were higher than that of Sham (P < 0.001). In open-field (OP) test, the central crossing number by OVX and Sham-LPS groups were lower than Sham (P < 0.001) while there were no significant differences between OVX-LPS and OVX groups. In elevated plus maze (EPM), the percent of entries to the open arm by both OVX and Sham-LPS groups was lower than that of Sham group (P < 0.001). The results of present study showed that deletion of ovarian hormones induced sickness behaviors in rats which were comparable to the effects of LPS. Moreover, further investigations are required in order to better understand the mechanism(s) involved.Neuroimmune factors have been proposed as the contributors to the pathogenesis of sickness behaviors. The effects of female gonadal hormones on both neuroinflammation and depression have also been well considered. In the present study, the capability of deletion of ovarian hormones to induce sickness-like behaviors in rats was compared with the effect lipopolysaccharide (LPS). The groups were including Sham, OVX, Sham-LPS, and OVX-LPS. The Sham-LPS and OVX-LPS groups were treated with LPS (250  The behbehavior . Proinflbehavior . Depressbehavior . Depressbehavior . In addibehavior . Depressbehavior , 6. Incrpression , 8. Thusα, IL-1, and IL-6 [α in glial cells in response to endotoxins [Ovarian hormones have been well known to have an important role in the regulation of mood and cognitive functions . They aland IL-6 . It has and IL-6 . It has dotoxins . A part dotoxins . It has dotoxins –14.α. Systemic injection of LPS causes spectrum behavior responses such as sickness behavior that animals show reduction in locomotor activity, exploration and feeding [Lipopolysaccharide (LPS), an endotoxin of gram-negative bacteria, is used for inducing chronic inflammation in the rodent . LPS-ind feeding . It also feeding –18. Rega ad libitum properly. Animal handling and all related procedures were approved by the Mashhad Medical University Committee on Animal Research. The animals were divided into four groups: (1) Sham (n = 20), (2) OVX (n = 10), (3) Sham-lipopolysaccharide , and (4) ovariectomized-lipopolysaccharide . In Sham and Sham-LPS groups, 9-10 animals which had proestrous phase were selected and used for the behavioral studies.Sixty female Wistar rats, 12 weeks old (240 ± 10 g), were used. The animals were housed in 4-5 per standard cages, at room temperature (22 ± 2°C) on a 12 h light/dark cycle. Food and water were availableμg/kg; i.p.) before behavioral tests. The animals of Sham and OVX groups received 1 mL/kg of saline instead of LPS. Ketamine was purchased from Alfasan Company (The Netherlands). LPS was purchased from sigma .The animals in the Sham-LPS and OVX-LPS groups were treated by single injections of LPS  anesthesia. Anesthesia was confirmed by reduced respiratory rate and no response to gentle pinching of foot pad. Abdominal incision was made through the skin of the flank of the rats and ovaries and ovarian fats were removed. Ovaries were isolated by ligation of the most proximal portion of the oviduct before removal. The same procedure was performed on the sham rats except that the wound was closed without removing the ovaries . The aniIt was carried out in Sham and Sham-LPS groups to select the animals with proestrous stage for behavioral studies. The female rat estrous cycle is 4-5 days and includes 4 phases: (1) proestrous stage where estrogen levels are very high and its typical cell pattern is smears including primarily epithelial cells with large nuclei. (2) Estrous stage, typical cell pattern for this order, is smears containing primarily cornified epithelial cells. (3) Metaestrous stage comprises cornified cells, primarily cornified cells, and sometimes a few epithelial cells. (4) Diestrous with typical cell pattern consists of large numbers of leukocytes with scattered nucleated epithelial and cornified cells in smears. To ensure that the female rats were cycling, vaginal cytologies were started 1 week before each experience and continued every day. Rats were held in a nonstress position and quickly lavaged with approximately 1 mL of saline. Slides were read using light microscopy, and estrous categories were classified based on cytological characteristics , 21.μg/kg (Sham-LPS and OVX-LPS groups) or saline (Sham- and OVX groups) [All animals were compromised to the test room environment for 1 h before the beginning of the experiment. During the forced swimming test (FST), the animals were placed in a glass cylindrical tank with 60 cm height and 38 cm width which was filled with water (24 ± 1°C) to the depth of 40 cm. The water was changed between each animal. In the first day, the rats were placed inside the water cylinder for 15 min (pretest) and then the animals were placed individually inside the water cylinder for 5 min for the following three alternative days (test days) , 23. Two groups) , 23. Theμg/kg (Sham-LPS and OVX-LPS groups) or saline (Sham- and OVX groups) [Open-field test was carried out for studying the depression-like behaviors of the animals. In the present study, the open-field measurement was done by a Plexiglass apparatus with an area of 100 × 100 cm and height of 40 cm. The inside of the apparatus was divided into 16 equal squares using a black line. In addition, within the apparatus was divided to two zones called peripheral and central zones , 23. All groups) , 23.The elevated plus maze (EPM) was made of 4 arms (50 cm length × 10 cm width) elevated 100 cm above the floor. Two of the arms which are named closed arms had 40 cm high dark walls and the other two which are named open arms had 0.5 cm high ledges . The betP < 0.05.The data were expressed as mean ± SEM. Repeated measures ANOVA were run followed by Tukey post hoc comparisons test. The criterion for the statistical significance was P < 0.001; P < 0.01–P < 0.001; The immobility times in both the OVX and Sham-LPS groups were higher than that of the Sham group . As shown in P < 0.001). There were no significant differences between Sham-LPS and OVX group. The results also showed that the central zone crossed number by the animals of OVX-LPS group was not different form that of OVX group (P < 0.001). The animals of Sham-LPS group spent lower times and traveled lower distance in central zone compare to Sham group ; however, there were no significant differences between Sham-LPS and OVX group (P < 0.01–P < 0.001). The animals of Sham-LPS group spent lower times and traveled distance in the peripheral zone compare to Sham group . As shown in P < 0.001); however, there were no significant differences between Sham-LPS and OVX group. The total crossing number by the animals of OVX-LPS group also was lower than that of OVX group . The animals of Sham-LPS group traveled shorter distance compared to Sham group -γ in patients with cancer is accompanied with depressive symptoms [It has been previously reported that administration of LPS in doses lower than the doses used in the present study resulted in avoidance of the central area of the open-field by the rats , 31. Thesymptoms , 33. On symptoms . It has symptoms . For exasymptoms . Knockousymptoms . It has symptoms  which arsymptoms , 39. Theμg/kg and 10 μg/kg LPS to substantia nigra induced an anxiety-like behavior in rats which was presented by decreasing in the percentage of the time spent in the open arms, the number of open-arm entries and the number of crossing in elevated plus maze [It has also been reported that LPS activates the hypothalamic-pituitary-adrenal axis thus increasing adrenocorticotropic hormone (ACTH) and glucocorticoids . In addilus maze . In the Ovarian hormones not only play an important role in reproductive behavior but are also involved in emotion, memory, neuronal survival, and the perception of somatosensory stimuli , 43. ResThe results of present study also showed exploratory behaviors in open-field test were decreased in both OVX and Sham-LPS groups compared to the Sham group. These data confirm the similar capability of ovarian hormones deletion and inflammation induced by LPS to induce sickness behaviors and depression.A large number of evidence show that LPS , 37 and It has been reported that anxiety-like behavior occurred in OVX rats when the animals were examined in the elevated plus maze test . ClinicaEPM is a well-known test which has been frequently used for evaluation of anxiety like behaviors in which the percent of the time in open arms is compared between the groups , 41, 55.In conclusion, the results of present study using forced swimming, open field, and elevated plus maze tests showed that deletion of ovarian hormones induced anxiety and depression like behaviors which might be considered as a sickness behavior in rats. These behaviors were comparable to the effects of lipopolysaccharide. Therefore, some common mechanism(s) might be suggested; however, it needs to be more investigated in the future.